Determination and dietary exposure assessment of 79 pesticide residues in Chinese onion (Allium fistulosum L.)

ABSTRACT Chinese onion is a common vegetable in daily diet, especially in Southwest China. We collected 184 samples from farm and market in Chongqing Municipality during 2020–2021 and established a multi-residue method for analysis of 79 pesticide residues on Chinese onion. Sixty-three of 184 samples were found pesticide resdues, accounting for 34.2%. A total of 31 pesticides were detected, and 6 pesticides in 11 samples have exceeded the Chinese MRLs, 5.98% of all samples. The chronic and acute dietary exposure assessment showed the %ADI and the %ARfD value were 0.005%–5.46% and 0.03%–297.95%, respectively. The %ARfD of most pesticides were acceptable except for cyhalothrin (297.95%, children; 140.17%, adults) and carbofuran (107.94%, children). The study indicated that pesticide residues on Chinese onion had different concentration levels which were low commonly. The establishment of MRLs for several pesticides (cyfluthrin, carbendazim, propamocarb) in Chinese onion is required for better estimation of dietary risk.


Introduction
Pesticide is a key agricultural input that can help to protect crops from the plant pests and diseases and increases crop yield.However, pesticides also have negative environmental impacts through contamination of soil, water, and nontarget plants and animals that can decrease biodiversity and, in some cases, affect human health (Aktar et al., 2009;Tudi et al., 2021).China is the largest producer and the highest application of pesticides in the world.In 2019, China's pesticide use reached 1.77 million tons, or about 13.07 kg/ha (FAO, 2022).Pesticide residues in vegetables and fruits have become one of the most common food safety issues around the world (Philippe et al., 2021;Syed et al., 2014;Tripathy et al., 2022;Zhou & Jin, 2009).Monitoring pesticides residue level on agro-products and assessing its risk were conducted commonly by researchers in many countries (Chu et al., 2020;Elgueta et al., 2021;Poulsen et al., 2017).
The vegetable of genus Allium is a popular and healthy food in the world, and the family has more than 900 species distributed in the Northern Hemisphere (Ekşi et al., 2020).Chinese onion (Allium fistulosum L.), also commonly called bunching onion, long green onion, and spring onion, is an edible species of Allium, native to China and cultivated in many other Asian countries (ITIS, 2010;PROTA4U, 2021), and the annual Chinese onion production in China reached 0.94 million tons, ranking first in the world in 2020 according to the FAO (2020).Chinese onion is very common in the daily diet in China and other Asian countries and can be used as a main dish, or a side dish and seasoning.Their extracts were also applied in medicine or health care (Aoyama et al., 2008;Chen et al., 1999;Lee et al., 2012;Sung et al., 2011).Some allium species (Allium cepa) can be used as a model bioassay to evaluate pesticide toxicity (Camilo-Cotrim et al., 2022).With the large-scale planting of Chinese onion, the occurrence of diseases and pests is inevitable, they are affected by some diseases and pests (Ettiene et al., 2006).It is important to control these diseases and pests by applying pesticides correctly.But the fact remains that some growers used pesticide improperly is the main reason of pesticide residues on vegetables (Zhou & Jin, 2009).Vegetables are one of the main foods that people eat every day, and pesticide residues in vegetables pose a serious risk to consumers.Studies have confirmed that long-term exposure to pesticides could have chronic impacts on health and lead to increased risks of illness, such as asthma, Type 2 Diabetes, Parkinson's disease, leukemia, colon cancer, cognitive effects, etc. (Evangelou et al., 2016;Kim et al., 2017;Martin et al., 2018;Parrón et al., 2011;Van Maele-Fabry et al., 2019).A study indicated that a high proportion of neurological health symptoms and cholinesterase test depression were observed among pesticide applicators (Mwabulambo et al., 2018).It is necessary to develop methods to detect pesticide-residues in genus Allium vegetables.Analysis methods of pesticide-residues for the genus Allium vegetables have been reported in some references, these reported methods, which sample extracts were cleaned by d-SPE (disperse-Solid Phase Extraction) and detected with GC (gas chromatography) (Ueno et al., 2003), GC-MS (gas chromatography-mass spectrometry) (Wang et al., 2017), and LC-MS/MS (liquid chromatography-tandem mass spectrometry) (Guan et al., 2014;Rodrigues et al., 2010Rodrigues et al., , 2012)).To assess the risk for dietary exposure to pesticide residues on Chinese onion, assessment methods could be employed, and the methods commonly consist of acute exposure assessment and chronic exposure assessment, and achieved by point assessment (Wang et al., 2022;Yuan et al., 2014).
In this study, the data of pesticide residues on Chinese onion were determined by GC-MS/MS (gas chromatographytandem mass spectrometry) and LC-MS/MS and analysed by the compliance with the Chinese MRLs (maximum residue levels) and point assessment.The aim of this study was to analyse pesticide residues and provide a corresponding dietary exposure assessment considering acute and chronic exposures for Chinese onion in local farms from the Chongqing Municipality in southwest China.

Chemicals, reagents and instruments
All pesticide standards were purchased from ANPEL Laboratory Technologies Inc. (Shanghai, China).HPLC grade methanol, ethyl acetate and acetonitrile were obtained from ThermoFisher (USA).Extraction and purification Kit was purchased from Agilent Technologies (China).Ultra-pure water was made by a MilliQ UF-Plus system (Millipore, Germany) with a resistivity of at least 18.2 MΩ.cm at 25°C.Needle-type membrane (0.22 μm) filters were purchased from Tianjin Jinteng Experimental Co., Ltd.

Preparation of standard solution
The concentration of each packed pesticide standard was 1000 mg/L in an ampere bottle.These packed pesticide standards were diluted, respectively, to 100 mg/L standard stock solutions, with different solvents according to their solubilities.HPLC grade acetone and acetonitrile were used to dilute the 49 pesticides for GC -MS/MS analysis and 30 pesticides for LC-MS/MS analysis (see Table S1), to a 2-4 mg/ L mixed standard working solution.Standard stock solutions and standard working solutions were stored at 4°C and −18°C, respectively, and keeping away from light.

Sample collection
Samples from different seasons were randomly taken at retailers or farms in different parts of Chongqing Municipality.From 2020 to 2021, a total 184 samples of Chinese onion were collected, including 93 samples in 2020 and 91 samples in 2021.Samples were taken according to guidelines in China (SAC, 2021), and the sample size was at least 1 kg.Samples were immediately wrapped in aluminum foil and chopped in little slice, blended in mushy, and placed in a freezer at −18°C, tested in the laboratory as soon as possible.

Sample preparation
The steps of extraction and purification method were in accordance with the Chinese standard method (SAC, 2008(SAC, , 2018)).The only difference was that this method achieved an extraction and purification method, which can be used for GC-MS/MS and LC-MS/MS on-line detection at the same time.The detailed steps were as follow: the 10 g of homogenized sample was weighed into a 50 mL plastic centrifuge tube, and added successively with 10 mL of acetonitrile, 4 g of magnesium sulfate, 1 g of sodium chloride, 1 g of sodium citrate and 0.5 g of disodium hydrogen citrate, covered the centrifuge tube with the cap, shaked vigorously for 1 min, and then centrifuged at 4200 rpm for 5 min.A purification step was needed as the following: 6 ml supernatant was pipetted into 15 ml plastic centrifuge tube containing 900 mg magnesium sulfate and 150 mg PSA, centrifuged at 4200 rpm for 5 min.For GC-MS/MS, transferred accurately 1 ml supernatant into 10 ml glass tube, and evaporated to near dryness with nitrogen at 40°C water bath, and reconstituted with 1 ml ethyl acetate, passed through the 0.22-μm filter for determination.For LC-MS/MS, 0.5 mL of the upper acetonitrile phase was mixed with 0.5 mL of a methanol/water (V/V, 1/1) solution, and the mixture passed through the 0.22-μm filter for determination later.The spiking experiments were evaluated to access the recovery and precision of the analytical method.Three concentration levels were investigated by six repeated determinations for each concentration level in one day.
The GC column oven temperature program is as follows: initially, set at 60°C held for 1 min, 30°C/min rate to 150°C, then 10°C/min rate to 300°C for 5 min holding time.Helium (99.999% purity) was used as carrier gas with a constant flow rate of 1.2 mL/min, and collision gas was argon (99.999% purity).MS parameters, such as transfer-line temperature (280°C) and ion source temperature (250°C), were maintained.Samples were ionized in MS/MS by the positive electron impact (EI) mode using an electron energy of −70 eV.The MRM scan mode was selected and two MRM transitions per analyte were monitored, as listed in Table S1.The solvent delay was fixed at 3 min.The splitless injection mode with the volume of 1 μL, surge pressure of 250 kpa and an injector temperature of 250°C was maintained.The total time program ran within 24 min.
For the LC part, the mobile phase A was 5 mmol/L formic acid-ammonium acetate aqueous solution, and the mobile phase B was methanol.Elution was performed in the gradient mode [time 0-1 min, 60% A; 1-7.5 min, 60-5% A, holding 1.5 min; 7.5-8.5 min, 5-60% A] and total analysis time of 8.5 min.The conditions were a flow rate of 0.2 mL/ min, an injection volume of 2 μL and a column temperature of 40°C.The ion source of MS was electron spray ionization (ESI), with an ion source interface voltage: ESI+/ESI-of −3.5 kV/3.5 kV, and the source temperature was 120°C.The desolvation gas temperature was set at 500°C with nitrogen flow rate of 800 L/h.The collision gas (argon) flow rate was set at 0.15 mL/min.All the parameters for MRM transitions and collision energy were optimised in order to obtain highest sensitivity and resolution (Table S2).

Estimation of the dietary exposure assessment
The dietary exposure assessment method of pesticide residues usually adopted deterministic estimate (also named point estimate), was established by JMPR (Joint FAO-WHO Meeting of Pesticide Residues) in 1995 (FAO/WHO, 1995), which definition referred to a brief description of consumer exposure parameters in the form of point values, and was widely used internationally and was also the mainstream model for the assessment of dietary exposure risk of pesticide residues in China.The model assumes that all individuals have the same consumption level of food, and there is a certain component in all kinds of food, which is measured by the same index.In this study, the children and general population were selected as mainly consumption group, and the dietary structure data of populations in China were from Survey report on nutrition and health status of Chinese Residents (Wang, 2008).According to the assessment needs, it was divided into chronic and acute dietary exposure assessment models.

Estimation of the chronic dietary exposure assessment
The method of NEDI (National Estimated Daily Intake) was used for chronic dietary exposure assessment (FAO, 2008; Ministry of Agriculture and Rural Affairs, 2015), and it is calculated according to the Supervised Trials Median Residue (STMR) or MRLs.The STMR value of each pesticide in Chinese onion should be derived from the supervised field trials, but obviously this value cannot be obtained in this study.All data of pesticide residues came from the monitoring of market products.Therefore, we used the mean pesticide residue level instead of STMR.The modified calculation formulas were Equations ( 1) and ( 2) (Chu et al., 2020;Nie Ji-Yun et al., 2014;Qian, 2007;Szpyrka et al., 2015).
NEDI -National Estimated Dietary Intake, mg/kg•d.STMR -Supervised trials median residue.Here, the STMR value was the mean pesticide residue level in Chinese onion samples based on monitoring data, mg/kg.F -The average daily consumption of Chinese onion, kg/d.ADI -The acceptable daily intake, mg/kg•d.bw -The average weight of body, kg.ADI% -The percentage of the national estimated daily intake in the allowable daily intake.When ADI% ≤100%, the risk of chronic dietary intake is considered acceptable; When ADI% >100%, the risk of chronic dietary intake is unacceptable.

Estimation of the acute dietary exposure assessment
Based on the dietary structure and specific food characteristics of each meal or day, the International Estimated Shortterm Intake (IESTI) is calculated with the STMR or HR (highest residue) for an acute exposure assessment, and calculations of the acute dietary exposure differ depending on different cases.FAO/WHO was divided into three cases about acute exposure assessment, Case 2a was suitable for Chinese onion acute exposure assessment (FAO/WHO, 2021).The IESTI and %ARfD was calculated by Equations ( 3) and (4) (Ministry of Agriculture and Rural Affairs, 2015; Nie Ji-Yun et al., 2014; Qian, 2007).
IESTI -International Estimated Short-term Intake, mg/kg•d.HR -Highest residue, the highest residual value for a pesticide from the monitoring data, mg/kg.
LP -Highest large portion reported (97.5 th percentile of eaters), kg/d.U e -Unit weight of the edible portion, kg.v -Variability factor, the factor applied to the composite residue to estimate the residue level in a high-residue unit, and the value is 3 recommended by FAO/WHO for Chinese onion.
bw -The average body weight, kg.ARfD -The acute reference dose, mg/kg.%ARfD -The percentage of acute reference dose.When %ARfD ≤100%, the acute risk is acceptable; When %ARfD >100%, it represents an unacceptable risk for dietary exposure.

Quality assurance of method
All the pesticides reported in this study were previously validated following the guidelines of SANTE 12682/2019, which include linearity, precision (relative standard deviation, RSD), recovery, and limit of quantification (LOQ) parameters.The results of linear equation, linear range, correlation coefficient, recovery results and LOQ are shown in Table S3.The values range of recovery, RSD and LOQ were 68.8-108.7%,0.6-10.6%,and 0.01 mg/kg, respectively.These results were proven to be a good accuracy and precision method for determining the levels of 79 pesticides by GC-MS /MS and LC-MS/MS on Chinese onion.

Pesticide residues in Chinese onion
A total of 79 pesticides were selected as targets, according to the Chinese Standards and Farming Investigation.The residual levels of 79 pesticides on 184 samples were analyzed, such as mean value, detection range, and median value, etc.The results were shown in Table 1.
In 2020-2021, 184 unprocessed samples were collected from market in Chongqing, and tested in laboratory.Pesticide residues were found in 63 of 184 samples, and the detection rate was 34.2%.Among the detected samples, 11 of 63 samples (17.5% of detected samples) have exceeded Chinese MRLs, accounting for about 6% of all samples, and 6 pesticides were detected in the 11 samples.In terms of years, the total detection rate of samples in 2020 and 2021 was 34.4% and 34.1%, and the exceeding rate was 9.7% and 2.2%, respectively.
A total of 31 different pesticides were detected in the Chinese onion (Table 1).In these pesticide residues, 18 insecticides, 12 fungicides, and 1 plant growth regulators were found.Moreover, the data also indicated that the detection frequency of fungicides was higher than that of insecticides, 108 and 91 times, respectively; at the same time, and the detection concentration of fungicide was also higher than insecticide.
Single-pesticide residue was detected in 27 samples (14.7%), and multiple-pesticide residues were detected in 36 samples (19.6% of all samples) (Figure 1).Monitoring data indicated that types of pesticide residue on Chinese onion were similar and samples with multiple residues on a single sample were typical (Figure 1), even 14 pesticides were found in a single sample.In all detected samples, the order of detected pesticide types from high to low were insecticides, fungicides and plant growth regulators.However, when the pesticide types of detection frequency was above 10, the order from high to low was fungicides and insecticides.Pesticides exceeding Chinese MRLs were emamectin benzoate (0.073 mg/kg), isofenphos-methyl (0.246 mg/kg, 1.202 mg/kg), chlopyrifos (0.35 mg/kg), carbofuran (0.049 mg/kg), cyhalothrin (0.244 mg/kg, 0.270 mg/kg, 2.705 mg/ kg) and thiamethoxam (0.381 mg/kg, 0.356 mg/kg, 0.451 mg/kg), and they were all insecticides, no fungicides.Among those pesticides, chlorpyrifos, carbofuran and isofenphos-methyl were prohibited using on vegetables according to Chinese national standards (SAC, 2021).

Chronic dietary exposure assessment
The risk factor ADI values were invoked from those specified in the Chinese national standard (SAC, 2021).The per capita daily consumption of Chinese onion (F) was 0.012 kg/d; The average weight of Chinese children aged 3-5 years was 18 kg (Chang & Wang, 2016).The average weight of general population (bw) was 53 kg, according to the ESTI table (FAO/WHO, 2021).In our study, STMR represented the average concentration level of pesticide residue in samples collected from the market.Seven pesticides (cyfluthrin, profenofos, emamectin benzoate, triazophos, isocarbophos, deltamethrin and phoxim) were detected at one frequency, 3 of 7 pesticides (cyfluthrin, Profenofos, and deltamethrin) did not have any MRL information on Chinese onion or bulb vegetables, so the STMR value could not be determined.Therefore, replacing the STMR value of 7 pesticides with the MRL if it has, according to the principle of dietary exposure assessment.The results of chronic dietary exposure of 28 pesticide residues detected on Chinese onion are shown in Table 2.The % ADI values of 28 residual pesticides were 0.01-16.09%for children and 0.005-5.46%for adults, and the %ADI values of each residual pesticide were far less than 100%, which indicated that the risk of chronic dietary intake of residual pesticides in Chinese onion was acceptable to the children and adults population.

Acute dietary exposure assessment
The acute dietary exposure assessment was carried out only for the types of pesticides that can be retrieved at present.The Food and Agriculture Organization (FAO) has noted it unnecessary to set acute reference doses for azoxystrobin, pyridaben and pyrimethanil, and that the ARfD value of some pesticides cannot be determined, such as isocarbophos, phoxim, isofenphos-methyl, paclobutyl, chlorochlor and chlor.Therefore, a total of 22 pesticides were assessed.The Chinese onion LP was 0.13653 kg/d (children) and 0.28928 kg/d (adults), and U e was 0.130 kg (FAO/WHO, 2021).The risk assessment results of acute dietary exposure of the remaining 22 pesticides were shown in Table 3.The % ARfD values of 22 residual pesticides ranged from 0.06% to 297.95% for children, and 0.03% to 140.17% for adults.Cyhalothrin had the highest %ARfD value, which were 297.95% and 140.17% for children and adults, respectively.Secondly, the %ARfD of carbofuran were 107.94% and 50.78%, respectively, for children and adults.According to the principle of dietary exposure assessment, the %ARfD value of cyhalothrin for children and adults, carbofuran for children, were more than 100%, and that means there was an unacceptable risk for children and adults in theory.Since cyhalothrin and carbofuran both have exceeded the MRLs, and cyhalothrin has a very high HR value (2.705 mg/kg) and using carbofuran in vegetables was prohibited, it was easy to result in %ARfD >100%.We must pay more attention to these two pesticides, using them scientifically and rationally in order to avoid the potential threat to our health.Except for the %ARfD values of cyhalothrin and carbofuran, all investigated pesticides were less than 100% for all groups, which showed that the risk of acute dietary intake of residual pesticides in Chinese onion was safety to the general population.

Discussion
The consumption of onion in the world is large, and there are many investigations and reports on pesticide residues, which were found in onion less frequently and less exceedances of MRLs (EFSA & Medina Pastor, 2021, 2022).The production and consumption of Chinese onion (Allium fistulosum L.) are smaller than those of onions (Allium cepa).
Currently, China is a major producer and consumer of Chinese onion.However, the study on dietary exposure assessment of pesticide residues in Chinese onions has not been reported.In our study, a total of 11 samples have exceeded the Chinese MRLs, refer to six pesticides, which mentioned in section 3.2.Among these pesticides, which have exceeded the Chinese MRLs, three pesticides were prohibited using on vegetables, accounting for 50% of the 6 pesticides and 27.3% of the 11 samples.A high rate of exceeding the Chinese MRLs was caused by some pesticides that had no specific MRL on Chinese onion, such as isofenphos-methyl, chlorpyrifos, and cyhalothrin, and the MRLs of these pesticides on bulb vegetables were very low, Chinese onion belongs to the bulb vegetable.As part of our study, the investigation by the Hong Kong Food Safety Center also found that cyhalothrin (0.35 mg/kg) in Chinese onion exceeded MRL (0.2 mg/kg) (HKFS, 2015).Some pesticides had a high detection concentration and no any corresponding residue limits about Chinese onion or bulb vegetables, such as cyfluthrin, carbendazim and propamocarb, and the highest detection concentration of these pesticides were 1.169 mg/kg, 3.600 mg/kg, and 3.100 mg/kg, respectively.But the three pesticides all have MRLs in onion/scallion in the EU, the U.S.A and Japan (CFR, 2022;Commission, 2021;Foundation, 2021), value of MRLs as follows: cyfluthrin (the EU 0.01 mg/kg, the U.S.A 0.01 mg/kg, Japan 2.0 mg/kg), carbendazim (the EU 0. 1 mg/kg, the U.S.A 0.1 mg/kg, Japan 3.0 mg/kg), propamocarb (the EU 30 mg/kg, the U.S.A 2.0 mg/ kg, Japan 3.0 mg/kg).Accordingly, we strongly recommended that government departments should formulate the MRLs for these pesticides on Chinese onion as soon as possible.
The %ADI of all evaluated pesticides were less than 100%, and the results of chronic dietary exposure assessment showed that they would not pose an adverse health to the general population.In the acute dietary exposure assessment, the %ARfD of most assessed pesticides were below 100%, but cyhalothrin was 297.95% in children and 140.17% in adults, and carbofuran was 107.94% for children.The % ARfD values of cyhalothrin and carbofuran indicated that the two pesticides have the risk of acute dietary exposure to children and adults (only cyhalothrin), and similar study results also found these two pesticides in Chinese onions (HKFS, 2015;Lv et al., 2022).It is suggested to strengthen the pesticide registration management of these two pesticides in Chinese onion.

Conclusions
The study shows that many pesticides were detected on Chinese onion samples, which collected from the region of Chongqing of China, and six pesticides in the 11 samples have exceeded the Chinese MRLs, accounting for 5.98% in total samples, and a banned pesticide (carbofuran) was detected in samples.Chinese onion consumption does not pose a danger to the health of children and adults based on our assessment for most investigated pesticides.
The %ADI value of all pesticides indicated that chronic dietary exposure was acceptable, and %ARfD for most of the pesticides detected in this study was lower than the standards of human health except for cyhalothrin and carbofuran.The %ARfD value of cyhalothrin was the highest in this study.A specific MRL of cyhalothrin should be formulated on Chinese onion according to this study.Moreover, some commonly used pesticides were found in monitoring, such as cyfluthrin, carbendazim and propamocarb, should be established MRLs in Chinese onion.In general, the results of this study indicated that it was inevitable to strengthen the monitoring of pesticide residues on Chinese onion and will provide a scientific basis for the government to formulate MRLs on a minor species vegetables.

Figure 1 .
Figure 1.Number of samples with one and multiple residue.

Table 1 .
Average concentration and percentage occurrence of detected residues in Chinese onion collected from Chongqing, China.
*Chinese MRLs of the bulb vegetables.−There was no value of China MRLs on Chinese onion or the bulb vegetables.ND -It indicates that there was no value of Mean and Median.Number-percentage -Number represents the detection times of specific pesticide; percentage is frequency of detection in samples.I -Insecticide; F -Fungicide; PGR -Plant growth regulator.

Table 2 .
Chronic dietary exposure assessment of pesticide residues in Chinese onions.

Table 3 .
Acute dietary exposure assessment of pesticide residues in Chinese onions.